The hepatotoxicity of acetaminophen is mediated by a chemically reactive metabolite which is formed in a minor P-450-dependent pathway in the target cells. However, species difference (and in man probably inter-individual difference) in susceptibility depends largely on the activity of the pathways leading to the glucuronide and sulfate conjugates of the drug. These conjugative pathways are the major routes of elimination and determine the mean residence time of the drug in the body: the greater the residence time, the greater is the amount of reactive metabolite formed and the greater is the hepatic injury. Conversely, the greater the activity of the conjugative pathways, the shorter the residence time and the less the hepatic injury. We have found that the resistance of diabetic rats to acetaminophen hepatotoxicity is due largely to their increased activity of the conjugative pathways of drug elimination; and that this increased activity is due in turn to increased capacities to synthesize the cosubstrates for conjugation, UDPGA and PAPS. Present evidence indicates that the regulation of synthesis of UDPGA and PAPS resides in the normal pathways of intermediary carbohydrate and protein metabolism of the liver cell. Thus, susceptibility is intimately dependent on the biochemical homeostasis of the liver cell and subject to influence by factors (nutrition, morbidity) which alter this homeostasis. We propose to identify and characterize the processes which regulate the formation of hepatic UDPGA and PAPS during the metabolism of hepatotoxic doses of acetaminophen. Experimentally, we will alter demand for hepatic UDPGA and PAPS by varying the dose of acetaminophen in animals which have altered ability to respond to this demand. Animal response will be modulated by physiological (fasted vs fed). and pathophysiological (normal vs diabetic) factors, and by drug metabolism inducers (phenobarbital, 3-MC, PCN), redox modulators (lactate, alanine, ethanol, dichloroacetate) and carbohydrate source (glucose vs galactose, fructose). Response will be determined in terms of acetaminophen metabolism/hepatotoxicity and by measurement of effects on levels of key enzymes, intermediates, UDPGA and PAPS, and by flux studies. For PAPS, the half-life of 35-S-cysteine and 35-SO-4 will also be determined. Additional studies on the control of hepatic GSH will focus on the roles of GSH synthesis and breakdown in the regulation of GSH levels in normal vs diabetic rats and in fed vs fasted rats and hamsters.